The focus of this proposal is to identify and characterize protein domains necessary for the assembly of large-conductance KCa channels. The minimal functional KCa channel is a homotetramer. Individual subunits must incorporate into the membrane and fold properly, and these subunits then specifically identify, and associate with, like subunits. While KCa channel assembly has not been studied, the assembly of other membrane protein complexes has been examined in some detail, and this knowledge will be used as a guide for the experiments outlined in this proposal. To begin to understand KCa subunit association, the domain(s) necessary for this association will be identified, both functionally and biochemically. Once domain(s) are identified, the question of how association occurs will be addressed by structure-function experiments of the association domain(s). For example, putative electrostatic interaction will be disrupted, and then the effect of association at the functional and biochemical levels will be examined. The identification and characterization of the association domain(s) of KCa channels will provide general information of ion channel assembly, as well as specific information on KCa currents in the CNS.